The present invention relates to drug discovery, and more particularly to a strategy to clone drugs for G protein coupled receptors.
Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
It has been estimated that more than 50% of the drugs in clinical use today are directed at G protein coupled receptors (GPCRs). Small peptides can activate a number of receptors of this family, such as receptors for thyrotropin-releasing hormone (TRH), which is a tripeptide (Gershengorn and Osman 1996), thrombin, for which a hexapeptide is a full agonist (Tapparelli et al. 1993), and formyl-Met-Leu-Phe, which is a tetrapeptide (Perez et al. 1994). Small molecules can inactivate constitutively active GPCRs, such as benzodiazepines, which inactivate TRH receptor mutants that are constitutively active (Heinflink et al. 1995)(a constitutively active receptor is one that signals in the absence of agonist).
It appears that these small molecules interact primarily, if not exclusively, with the transmembrane (TM) bundle or extracellular (EC) loops of GPCRs (Cascieri et al. 1995). For example, it appears that the xe2x80x9cactivation domainxe2x80x9d of a GPCR with a large EC amino terminus, such as the receptor for calcitonin, is present within the region of the receptor from the beginning of TM helix one to the C-terminus, which includes the TM bundle and EC loops (Stroop et al. 1995).
The discovery of peptides that could activate GPCRs or inactivate constitutively active GPCRs may have enormous potential for clinical applications because a number of peptide agonists of GPCRs are currently used therapeutically and diagnostically. In the shorter term, the discovery of such peptides will yield reagents that could be used by pharmaceutical companies to identify ligands for or functions of xe2x80x9corphanxe2x80x9d receptors.
To this end, it is an object of the subject invention to provide a strategy to discover small peptides that will activate any G protein-coupled receptor (GPCR) or inactivate any constitutively active GPCR. These peptides could serve as lead chemicals for design of clinically useful drugs or could be used to identify the natural ligand or physiologic function of xe2x80x9corphanxe2x80x9d receptors, that is, putative receptors that have been identified (i.e., cloned) but for which the function is unknown. The strategy uses combinatorial peptide libraries tethered to the GPCR. With this approach, millions of random peptides of a given length can be tested for activity in the context of a library and those that activate GPCRs or inactivate constitutively active GPCRs can be identified.
The invention thus provides a method of identifying peptide agonists or negative antagonists of a G protein coupled receptor of interest. The method comprises expressing a peptide of a peptide library tethered to a G protein coupled receptor of interest in a cell, and monitoring the cell to determine whether the peptide is an agonist or negative antagonist of the G protein coupled receptor of interest.
In one embodiment for identifying peptide agonists, the expression of a peptide of a peptide library tethered to a G protein coupled receptor of interest in a cell comprises preparing a G protein coupled receptor construct, introducing the G protein coupled receptor construct into a cell, allowing the cell to express the G protein coupled receptor encoded thereby, and exposing the cell to a ligand of a self-activating receptor, wherein the ligand cleaves the G protein coupled receptor construct so as to expose the inserted peptide of the peptide library. The G protein coupled receptor construct for identifying a peptide agonist, which is also provided by the subject invention, comprises a nucleic acid molecule encoding a G protein coupled receptor with a deleted first amino terminus; a nucleic acid molecule encoding a second amino terminus of a self-activating receptor attached to the nucleic acid molecule encoding the G protein coupled receptor at the deleted first amino terminus, the second amino terminus having a deleted portion which is a peptide agonist for activating the self-activating receptor; and a nucleic acid molecule encoding the peptide of the peptide library inserted into the second amino terminus and replacing the deleted portion.
In a further embodiment for identifying peptide negative antagonists, the G protein coupled receptor of interest is a constitutively active G protein coupled receptor and the expression of a peptide of a peptide library tethered to the G protein coupled receptor of interest in a cell comprises preparing a constitutively active G protein coupled receptor construct, introducing the constitutively active G protein coupled receptor construct into a cell, and allowing the cell to express the constitutively active G protein coupled receptor encoded thereby. The constitutively active G protein coupled receptor construct for identifying a peptide negative antagonist, which is also provided by the subject invention, comprises a nucleic acid molecule encoding a constitutively active G protein coupled receptor with a deleted first amino terminus; a nucleic acid molecule encoding a second amino terminus of a self-activating receptor attached to the nucleic acid molecule encoding the constitutively active G protein coupled receptor at the deleted first amino terminus, the second amino terminus having a deleted portion which includes a peptide agonist for activating the self-activating receptor as well as any amino acids positioned amino terminally to the peptide agonist; and a nucleic acid molecule encoding the peptide of the peptide library inserted into the second amino terminus and replacing the deleted portion.
In a still further embodiment for identifying peptide agonists, the expression of a peptide of a peptide library tethered to a G protein coupled receptor of interest in a cell comprises preparing a G protein coupled receptor construct, introducing the G protein coupled receptor construct into a cell, and allowing the cell the express the G protein coupled receptor encoded thereby. The G protein coupled receptor construct for identifying a peptide agonist, which is also provided by the subject invention, comprises a nucleic acid molecule encoding a G protein coupled receptor with a deleted first amino terminus; a nucleic acid molecule encoding a second amino terminus of a self-activating receptor attached to the nucleic acid molecule encoding the G protein coupled receptor at the deleted first amino terminus, the second amino terminus having a deleted portion which includes a peptide agonist for activating the self-activating receptor as well as any amino acids positioned amino terminally to the peptide agonist; and a nucleic acid molecule encoding the peptide of the peptide library inserted into the second amino terminus and replacing the deleted portion.